Ribosomal protein S6 kinase 1 (S6K1), a critical mediator of cell growth, is overexpressed in breast cancer and is associated with poor prognosis. S6K1 has two known isoforms, p85(S6K1) and p70(S6K1). p85(S6K1) is characterized by 23 additional amino acids in the N-terminus of p70(S6K1). This is thought to target p85(S6K1) to the nucleus, while p70(S6K1) is mainly cytoplasmic. We sought to determine the activation, regulation, and function of p70(S6K1) and p85(S6K1) in breast cancer. We found that most breast cancer cell lines expressed both isoforms. Mitogen-dependent pathways concordantly regulated phosphorylation on T389, S371, and T421/S424. Phosphorylation of both isoforms was inhibited by PI3K/mTOR inhibitors. Mitogen-dependent pathways concordantly regulated the phosphorylation of the two isoforms on T389, S371, and T421/S424. Both isoforms had S6 kinase activity. We also detected a p60 isoform with antibodies to the p70(S6K1) C-terminal but not the N-terminal. Further studies on S6K1 isoforms are warranted for therapeutically targeting this pathway.